Gelatin–epigallocatechin gallate nanoparticles with hyaluronic acid decoration as eye drops can treat rabbit dry-eye syndrome effectively via inflammatory relief

Abstract

Introduction

Dry-eye syndrome (DES) is a general eye disease. Eye drops are the common ophthalmological medication. However, the ocular barrier makes it difficult to attain high drug bioavailability. Nanomedicine is a promising alternative treatment for ocular diseases and may increase drug content in the affected eye.

Methods

To explore this potential, we constructed nanoparticles (NPs) containing an anti-inflammatory agent for DES treatment. The NPs were made of gelatin–epigallocatechin gallate (EGCG) with surface decoration by hyaluronic acid (HA) and designated “GEH”. The particle size, surface charge, and morphology were evaluated. The in vitro biocompatibility and anti-inflammation effect of nanoparticles were assayed via culturing with human corneal epithelium cells (HCECs) and in vivo therapeutic effect was examined in a DES rabbit’s model.

Results

The synthesized GEH NPs had a diameter of approximately 250 nm and were positively charged. A coculture experiment revealed that 20 µg/mL GEH was not cytotoxic to HCECs and that an EGCG concentration of 0.2 µg/mL downregulated the gene expression of IL1B and IL6 in inflamed HCECs. Large amounts of GEH NPs accumulated in the cytoplasm of HCECs and the ocular surfaces of rats and rabbits, indicating the advantage of GEH NPs for ocular delivery of medication. Twice-daily topical treatment with GEH NPs was performed in a rabbit model of DES. The ocular surface of GEH-treated rabbits displayed normal corneal architecture with no notable changes in inflammatory cytokine levels in the cornea lysate. The treatment improved associated clinical signs, such as tear secretion, and fluorescein staining recovered.

Conclusion

We successfully produced GEH NPs with high affinity for HCECs and animal eyes. The treatment can be delivered as eye drops, which retain the drug on the ocular surface for a longer time. Ocular inflammation was effectively inhibited in DES rabbits. Therefore, GEH NPs are potentially valuable as a new therapeutic agent delivered in eye drops for treating DES.

Keywords:

• gelatin nanoparticles
• epigallocatechin gallate
• EGCG
• hyaluronic acid
• HA
• dry-eye syndrome
• DES
• anti-inflammation

Supplementary material

Total RNA extraction

After human corneal epithelium cells (HCECs) had been treated with variant epigallocatechin gallate (EGCG) formulations at certain times, cells were trypsinized and collected into a 1.5 mL Eppendorf tube. Trizol (1 ml) was added to the tube, left at 4°C for 1 hour, then centrifuged at 14,000 rpm for 30 minutes at 4°C. Chloroform (200 µL) was added and the tube shaken vigorously by vortex for 1 minute, then put on ice for 3 minutes. This protocol was repeated four times and the tube put on ice for 10 minutes and then centrifuged. The supernatant was transferred to the other tube, isopropanol added, the tube gently shaken, then kept on ice for 10 minutes. After centrifugation at 14,000 rpm for 30 minutes (4°C), the supernatant was removed and 1 mL 75% alcohol added, allowed to react for 3 minutes, and then centrifuged again. The supernatant was removed and left alone for 5 minutes to evaporate the residual ethanol. RNase-free water (10 µL) was added to dissolve the RNA. RNA concentration was then calculated as the ratio of absorbance at A₂₆₀:A₂₈₀.

Preparation of cDNA from RNA by reverse transcription

Add 10 µL of 2X RT Master Mix to the strip tubes, then added different groups of RNA samples (10 µL), pipetting for several times, then centrifuged for 3 minutes. These samples were then put in the mechanism and setting the thermal cycle for reverse-transcription followed by the direction. The temperature was set as: first stage at 25°C for 10 minutes, second stage for 120 minutes at 37°C, third stage kept at 85°C for 5 minutes, and finally placed in a 4°C refrigerator for further use.

Real-time PCR

The 20 µL cDNA was mixed with 80 µL of nuclease-free H₂O to prepare a cDNA templating. Add 10 µL of Master Mix, 4 µL of Nuclease-free H₂O, 1 µL of Probe (IL1B, IL6, IL8, TNFA and GAPDH), and 5 µL of cDNA sample to the strip tubes. These samples were centrifuged for 3 minutes, and then put into the real-time LightCycler 96 PCR system (Hoffman-La Roche, Basel, Switzerland) for the reaction. Temperature settings were as follows: first stage, 95°C for 600 seconds, then the second stage at 95°C for 10 seconds plus 60°C for 30 seconds. The second stage was repeated for 45 cycles.

Acknowledgments

This study was supported by grants from the Ministry of Science and Technology, Taiwan (NSC 102–2221-E-038–007 and MOST 106–2628-E-038–001-MY3) and the Integrated Research Grant in Health and Medical Sciences from the National Health Research Institute, Taiwan (NHRI-EX105-10334EI).

Disclosure

The authors report no conflicts of interest in this work.